Human cytochrome P450 (P450) enzymes are membrane proteins that play key roles in the metabolism of hydrophobic foreign molecules. Such xenobiotic metabolism deactivates most drugs, but can transform prodrugs and procarcinogens into active or carcinogenic forms, respectively. Each of these P450 enzymes can bind a chemically diverse range of substrates, then accept an electron from NADPH-cytochrome P450 reductase, bind molecular oxygen, and finally accept a second electron to monooxygenate the substrate. The small heme protein cytochrome b5 can variably accelerate, inhibit, or have no effect on this catalysis. Thus to accomplish drug metabolism or procarcinogen activation, a P450 interacts with very chemically distinct small molecule substrates and with multiple protein partners. Understanding and being able to predict which diverse foreign compounds are recognized and how they are metabolized by different P450 enzymes is of great practical value not only in understanding the metabolism of existing drugs and other chemicals to which we are exposed, but also in guiding the development of new drugs and chemoprevention efforts. However, structural information about how many different human P450 proteins orient substrates, say for metabolism to a carcinogenic form vs. a noncarcinogenic form, and how reductase and b5 protein interactions regulate such catalysis is a gap in our knowledge. This prevents the effective use of potentially valuable anticancer agents, understanding of cancer initiation events, and ultimately improved prevention and treatment of diverse disease states. The applicant's long-term research goal is to understand the structure/function principles that control P450 catalysis, in order that this information can b exploited to improve human health. The objective of this proposal is to extend our structural knowledge across current boundaries by determining initial structures for important human P450 enzymes of clinical utility, examining clinically-important P450/ligand complexes, and probing the relationships between P450 and other proteins involved in catalysis. Aim 1 focuses on new X-ray structures of lung 1A1 and 2F enzymes with procarcinogens activated to initiate lung cancer, in addition to 2W1, a P450 that can be used to activate anticancer prodrugs selectively for colon cancer treatment. These membrane P450 enzymes share specific substrates among themselves and with the 2A13 and 2E1 enzymes recently studied, advancing the longer-term goal of identifying structural features responsible for partially overlapping, yet distinct metabolism. The second aim moves from P450/ligand interactions to human P450 interactions with reductase and b5 proteins, complexes for which no structures are currently available. P450/reductase and P450/b5 complexes will be defined in detail for select human P450 enzymes using X-ray crystallography, while NMR provides the opportunity to readily compare and contrast such interactions across multiple medically-relevant human P450 enzymes. Based on our current data and demonstrated capabilities in structural and functional analysis of human membrane P450 enzymes, this work is expected to provide advanced structure/function information for multiple human enzymes that can be directly applied to advance human health in multiple disease states, but with a focus on lung and colon cancers.